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Agitation Modules: Flexible Means to Accelerate Automated Freeze Substitution

Reipert, Siegfried; Goldammer, Helmuth; Richardson, Christine; Goldberg, Martin W.; Hawkins, Timothy J.; Hollergschwandtner, Elena; Kaufmann, Walter A.; Antreich, Sebastian; Stierhof, York-Dieter


Siegfried Reipert

Helmuth Goldammer

Christine Richardson

Elena Hollergschwandtner

Walter A. Kaufmann

Sebastian Antreich

York-Dieter Stierhof


For ultrafast fixation of biological samples to avoid artifacts, high-pressure freezing (HPF) followed by freeze substitution (FS) is preferred over chemical fixation at room temperature. After HPF, samples are maintained at low temperature during dehydration and fixation, while avoiding damaging recrystallization. This is a notoriously slow process. McDonald and Webb demonstrated, in 2011, that sample agitation during FS dramatically reduces the necessary time. Then, in 2015, we (H.G. and S.R.) introduced an agitation module into the cryochamber of an automated FS unit and demonstrated that the preparation of algae could be shortened from days to a couple of hours. We argued that variability in the processing, reproducibility, and safety issues are better addressed using automated FS units. For dissemination, we started low-cost manufacturing of agitation modules for two of the most widely used FS units, the Automatic Freeze Substitution Systems, AFS(1) and AFS2, from Leica Microsystems, using three dimensional (3D)-printing of the major components. To test them, several labs independently used the modules on a wide variety of specimens that had previously been processed by manual agitation, or without agitation. We demonstrate that automated processing with sample agitation saves time, increases flexibility with respect to sample requirements and protocols, and produces data of at least as good quality as other approaches.


Reipert, S., Goldammer, H., Richardson, C., Goldberg, M. W., Hawkins, T. J., Hollergschwandtner, E., …Stierhof, Y. (2018). Agitation Modules: Flexible Means to Accelerate Automated Freeze Substitution. Journal of Histochemistry & Cytochemistry, 66(12), 903-921.

Journal Article Type Article
Acceptance Date Jun 11, 2018
Online Publication Date Jul 3, 2018
Publication Date 2018-12
Deposit Date Jul 5, 2018
Journal Journal of Histochemistry & Cytochemistry
Print ISSN 0022-1554
Electronic ISSN 1551-5044
Publisher SAGE Publications
Peer Reviewed Peer Reviewed
Volume 66
Issue 12
Pages 903-921